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P53 Signaling Modulation of Cell Cycle Arrest and Viral Replication In Xu et al. Vet Res (2016) 47:120 DOI 10.1186/s13567-016-0403-4 RESEARCH ARTICLE Open Access p53 signaling modulation of cell cycle arrest and viral replication in porcine circovirus type 2 infection cells Dan Xu†, Qian Du†, Cong Han, Zengguo Wang, Xiujuan Zhang, Tongtong Wang, Xiaomin Zhao, Yong Huang* and Dewen Tong* Abstract Porcine circovirus type 2 (PCV2) is a ubiquitous pathogen in the swine industry worldwide. Previous studies have shown that PCV2 infection induces host cell apoptosis through up-regulation of p53. To further identify the regula- tory roles of p53 signaling in the process of PCV2 infection, we established p53 gene knockout PK15 cell lines using the genomic editor tool CRISPR/Cas9, and further investigated the roles of p53 in modulating the cell cycle and viral replication in this study. The results show that PCV2 infection induced obvious S phase accumulation in wild-type PK15 cells and a compromised S phase accumulation in the p53 gene mutation cells (813PK15p53m/m), but did not p53 / induce obvious S phase accumulation in the p53 gene knockout cells (148PK15 − −) compared with the respec- tive mock infection. PCV2 infection activated p53 signaling, up-regulated the expression of p21, Cyclin E, and down- regulated Cyclin A, CDK2. In p53 deficient cells, however, PCV2-induced changes in Cyclin A, CDK2, and Cyclin E were efficiently reversed to the basal levels. Detection of PCV2 replication showed decreased viral ORF1 genomic DNA in p533 / p53m/m p53 deficient cells (148PK15 − −) and p53 mutated cells (813PK15 ) compared with p53 wild-type cells after different synchronization treatment. Furthermore, PCV2 viral genomic DNA and Cap protein levels were higher in the cells released from S phase synchronized cells than in the cells released from the G0/G1 phase or G2/M phase-syn- chronized, or asynchronous cells after 18 h post-infection. Taken together, this study demonstrates that PCV2 infection induces S phase accumulation to favor viral replication in host cells through activation of the p53 pathway. Introduction However, the roles of p53 signaling in modulating cell PCV2, belonging to the family Circoviridae, is the main cycle and PCV2 replication has not been defined up to pathogen to cause porcine circovirus associated diseases date. (PCVAD) [1], posing a huge threat for world pig hus- Numbers of studies have broadened our understand- bandry [2]. As a tiny DNA virus, PCV2 infection requires ing of the roles of p53 signaling in the process of different host cells to provide necessary resources for replication virus infection and replication. For instance, Kaposi’s sar- themselves, thus disturbing a variety of cell signaling coma herpesvirus (KSHV) activates host p53 signal and pathways to modulate the host cell cycle, proliferation, induces G2 phase arrest to promote the onset of virus survival and death to facilitate their infection and repli- replication [6]. Prototype foamy virus (PFV) promotes cation [3, 4]. Among the signaling pathways, p53 signal- p53 level increase by knockdown of Pirh2 to contrib- ing is essential for control of quiescent cell entry into the ute to the latency of PFV infection [7]. Herpes simplex cell cycle, and regulating cellular DNA replication [5]. virus type 2 infection can phosphorylate p53 protein to induce the G0/G1 phase arrest [8]. PRRSV manipulates the host factors mdm2 and p53 via its Nsp1α to increase *Correspondence: [email protected]; [email protected] viral replication at the early stage of infection [9]. Indeed, † Dan Xu and Qian Du contributed equally to this work previous studies have shown that PCV2 ORF3 protein College of Veterinary Medicine, Northwest A&F University, 22 Xinong Rd, Yangling, Shaanxi 712100, People’s Republic of China specifically interacts with porcine ubiquitin E3 ligase © The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. Vet Res (2016) 47:120 Page 2 of 11 Pirh2 to promote p53 accumulation [10], playing an CRISPR/cas9 KO cell important role in PCV2 pathogenesis [11], which indi- Targeting sites in the p53 gene were selected using the cates the key role of p53 in the interaction of PCV2 and CRISPR program (Genome Engineering. Broad Institute the host. However, in-depth study of the roles of p53 Cambridge, MA, USA) Oligonucleotide pairs for the target signaling in the process of PCV2 was limited due to lack- sequences were annealed and the resulting fragments were ing of p53 deficient cell line in porcines. then cloned into the BsmB I sites of lentiCRISPRv2 plasmid In this study, with the help of the CRISPR/Cas9 system, (Addgene), and co-transfected into HEK293T cells with the we constructed p53 deficient and mutant porcine cell packaging plasmids psPAX2 (AddGene 12260) to generate lines, and further detected and compared the difference the lentivirus. 72 h after the transfection, the supernatant of cell cycle profiles and viral replication between the was collected after three cycles of frozen-thawed. Titers of p53 wild-type, p53 deficient and p53 mutant porcine cell the obtained lentivirus expressing the target sequences were lines. This study allows us to deeply explore and confirm determined by qPCR. Finally, the CRISPR/Cas9 mediated the roles of p53 signaling in modulating cell cycle and P53 knockout cells were selected from lentivirus infected PCV2 replication. PK15 cell lines that were cultured in puromycin (500 ng/ mL) DMEM medium for at least 14 days. Genomic DNA Materials and methods sequence from PK15 cells was determined using primers: Cells, virus and antibodies 148-F: 5′-GACTCCTGTTGTTCCCATCCA-3′; 148-R: Porcine kidney 15 (PK15) cells purchased from ATCC 5′-AGGGAGCCAGCAGTCAAATG-3′; 813-F: 5′-GGGA (CCL-33) were cultured in Dulbecco’s Modified Eagle’s CGGAACAGCTTTGAGGT-3′; 813-R: 5′-CTGTTGGCA Medium (Gibco BRL, Gaithersburg, MD, USA) supple- AATGCCCCAAA-3′. mented with 10% heat-inactivated fetal bovine serum (Thermo Scientific HyClone, Beijing, China), and incu- Cell synchronization bated at 37 °C in a 5% CO2 atmosphere incubator. The Cells synchronized in G1/G0 phase were obtained by PCV2 strains (GenBank No. EU366323) used in this serum starvation. PK-15 cells were cultured in serum- study were isolated and purified previously by our team free medium for 24 h or 48 h, and then cells were washed and stocked in our laboratory, the UV-inactivation was with PBS and plated in fresh media to start PCV2 incuba- performed by UV radiation of the virus for 45 min in the tion for 1 h and cultured in 2% FBS DMEM medium for hood. The anti-PCV2 Cap primary antibodies were pro- 18 or 24 h for later analysis. Double thymidine block was duced by our team [12, 13]. The primary monoclonal rab- used for early S phase synchronization. The cells were bit antibodies of p53, p21 and anti-BrdU were purchased treated for 12 h with 2 mM thymidine, after which cells from Cell Signaling (Cell Signaling Technology, Danvers, were washed and released into fresh media with MOI = 1 MA, USA). CDK2, Cyclin A and Cyclin E antibodies PCV2 virus then incubated for 1 h, and cultured in 2% were purchased from Santa Cruz Biotechnology (Santa FBS DMEM medium for 18 h. The cells were treated with Cruz, California, CA, USA). The monoclonal antibody 100 ng/mL nocodazole for 16 h until arrest at the G2/M of β-actin was purchased from sigma (Sigma-Aldrich, phase, then the cells were released by washing with PBS St. Louis, MO, USA). The FITC goat anti-mouse IgG was and plated in fresh media to start PCV2 incubation for purchased from BD Biosciences (BD, San Jose, CA, USA). 1 h and culture in 2% FBS DMEM medium for 18 h for later analysis. Cell cycle analysis The ratio of cells in each phase of the cell cycle was deter- Detection of virus replication mined by DNA content using propidium iodide (PI) The cells were seeded in culture plates at a density of staining followed by flow cytometric analysis. The cells 1 106 cells/well, and cultured to reach approximately 6 × plated at a density of 1 × 10 cells/flask were treated 60–70% confluence. PCV2 strains were used to infect the with the indicated Multiplicity of infection (MOI) of cells at a multiplicity of infection of 1. Viral ORF1 frag- PCV2 for the indicated times as described in the figure ments were determined in each of the PK15 cell lines legends. The cells were trypsinized, washed twice with using primer PCV-F: 5′-AGTACCGGGAGTGGTAGG ′ ′ ′ PBS, and fixed with 70% ice-cold ethanol at− 20 °C over- AG-3 ; PCV-R: 5 -GTTGAATTCTGGCCCTGCTC-3 . night. Fixed cells were washed with cold PBS and resus- The supernatant and the attached cells were collected pended with PI staining solution containing 50 mg/mL together to extract the DNA. PI (Sigma-Aldrich), 100 mg/mL RNase A (TIANGEN Biotech, Beijing, China), and incubated in the dark for BrdU incorporation assay 30 min. The samples were analyzed using a flow cytom- For labeling of S-phase cells, BrdU was added in mid- eter (Accuri™ C6, BD Biosciences, San Diego, CA, USA). log phase cells at a final concentration of 10 µM and Xu et al. Vet Res (2016) 47:120 Page 3 of 11 incubated for 1 h at 37 °C.
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